Muscodor species- Endophytes with
Biological Promise
Author: Gary A. Strobel
This is a postprint of an article that originally appeared in Phytochemistry Reviews on February
16, 2010. The final publication is available at Springer via
http://dx.doi.org/10.1007/s11101-010-9163-3.
Strobel, Gary. “Muscodor Species- Endophytes with Biological Promise.” Phytochemistry
Reviews 10, no. 2 (February 16, 2010): 165–172. doi: 10.1007/s11101-010-9163-3.
Made available through Montana State University’s ScholarWorks
scholarworks.montana.edu
Muscodor species- Endophytes with Biological Promise
Gary Strobel
Department of Plant Sciences
Montana State University
Bozeman, Montana, 59717
Email address: uplgs@montana.edu
Key words: Mycofumigation, Endophytes, Volatile antibiotics, Biological Control
1
Abstract
A novel fungal genus is described that produces extremely bioactive volatile organic
compounds (VOC’s). The initial fungal isolate was discovered as an endophyte in
Cinnamomum zeylanicum in a botanical garden in Honduras. This endophytic fungus was
named Muscodor albus because of its odor and its white color. This fungus produces a
mixture of VOC’s that are lethal to a wide variety of plant and human pathogenic fungi
and bacteria. It also is effective against nematodes and certain insects. The mixture of
VOC’s has been analyzed using GC/MS and consists primarily of various alcohols, acids,
esters, ketones, and lipids. Final verification of the identity of the VOC’s was carried out
by using artificial mixtures of the putatively identified compounds and showing that the
artificial mixture possessed the identical retention times and mass spectral qualities as
those of the fungal derived substances. Artificial mixtures of the available VOC’s
mimicked some but not all of the biological effects of the fungal VOC’s when tested
against a wide range of fungal and bacterial pathogens. Other species and isolates of this
genus have been found in various tropical forests in Australia, Bolivia, Ecuador, and
Thailand. The most recent discovery is Muscodor crispans whose VOCs are active
against many plant and human pathogens. Potential applications for “mycofumigation”
by members of the Muscodor genus are currently being investigated and include uses for
treating plant diseases, buildings, soils, agricultural produce and many more. This report
will describe how the fungus was discovered, identified, and found potentially useful to
agriculture, medicine and industry.
Key words; Endophytes, Mycofumigation, Volatile organic compounds, DNA,GC/MS
2
Microbes for humankind
Microorganisms have long served mankind by virtue of the myriad of the enzymes and
secondary products that they make [1]. Interestingly, however, is the fact that only a
relatively small number of microbes are used directly in human applications i.e. bread,
cheese, and alcoholic beverage making. It seems that a more comprehensive search of the
earth’s niches may reveal novel microbes having other direct utilities to human societies.
Of course, the advantages of direct use are numerous including reduced economic and
regulatory factors while at the same time successfully completing a useful task. The
diversity of microbial life is enormous and the niches in which microbes live is truly
amazing ranging from deep ocean sediments to the earth’s thermal pools [2]. A relatively
untapped source of microbial diversity is the world’s rainforests. Each plant supports a
suite of microorganisms known as endophytes [3]. These organisms cause no overt
symptoms on the plants in which they live [4]. Furthermore, since so little work on these
endophytes has been done it is suspected that untold numbers of novel fungal and
bacterial genera exist as plant-associated microbes [5]. The rationale for sampling
rainforest species is the fact that high plant biodiversity existing in the world’s rainforest
areas may also be accompanied by high microbial diversity [3, 6]. Thus, we have begun a
concerted search for novel endophytic microbes and the prospects that they may produce
novel bioactive products as well as processes that may prove useful at the organismal
level. This report concentrates mostly on the discovery of one novel endophytic fungal
genus – Muscodor and the systematic study of its VOC production and its future for
human use, biological discovery, and biological control.
3
The discovery of Muscodor albus
In the late 90’s I was on a collecting trip in the jungles near to the Caribbean coast of
Honduras. I had selected this area to visit because Central America is one of the world’s
“hot spots of biodiversity” [6]. One modestly sized tree, not native to the new world, was
introduced to me as Cinnamomum zeylanicum. Small limb specimens were taken and
placed in a plastic bag and brought back to Montana and the sample was processed
according to standard isolation procedures for endophytes [3]. However, we had been
plagued with microscopic phytophagous mites in the lab for many months. This is not
uncommon for labs in which plant materials are being brought on a regular basis. Mites
infest the bench tops and invade parafilm sealed Petri plates containing agar in which
they take up residence. Thus, in order to eliminate this persistent mite problem we
decided to place the Petri plates, with plant tissues, in a large plastic box having a firmly
fitting lid. This maneuver would make it difficult for the tiny animals to find their way
from the bench surfaces to the inside of the box. After a few days most plant specimens
had sported endophytic fungal growth. Eventually the plates were removed and the
individual hyphae transferred to fresh plates of potato dextrose agar. After a day or two of
incubation we noted that no transferred endophyte grew except one. Had the placement of
the endophytes in the large plastic box killed the endophytes by limiting oxygen
availability? Soon it became obvious that the one endophytic fungus (designated isolate
620) remaining alive was producing volatile organic compounds (VOCs). The hypothesis
that an endophyte can make VOCs having antibiotic activities was born. It was quickly
learned that although many wood inhabiting fungi make volatile substances, none of
these possessed the biological activity of isolate 620 [7]. However some early data
4
supported the observations that Trichoderma sp. produced some VOCs, however, with
only modest biological activity and no attempt was made to identify the VOCs of this
organism [8]. Later, another report on Trichoderma sp. showed the identity of the VOCs
and pointed out that inhibitory activity was associated with these compounds [9].
Muscodor albus and its VOCs
Isolate 620 is a sterile (not producing spores) endophytic fungus possessing
some interesting hyphal characteristics including coiling, ropyness, and some right angle
branching. The mycelia of the fungus on most media are whitish and suppressed. In fact,
the mycelia commonly make undulations on the agar surface. However, it has a tendency
to produce aerial discrete organized mycelia yielding what appear to be synemma,
however closer inspection reveals that they too are sterile. All attempts to initiate spore
formation have failed. Isolate 620 grows well on a number of agar media including potato
dextrose agar and other media containing plant extracts or various media made with
wood shavings supplemented with starch since it appears that a rich carbohydrate source
is critical to VOC production by this organism [10].
Therefore, in order to taxonomically characterize this organism, the partial regions of the
internal transcribed spacer region and the 5.8S of the nuclear ribosomal DNA operon
(ITS- 5.8 rDNA) were isolated, sequenced, and deposited in GenBank as AF 324336 and
AF 324337 [10]. It turns out that isolate 620 is unique but it has a 82-92% sequence
similarity to several Xylaria spp. [10]. The GC/MS analysis of the fungal VOCs showed
the presence of at least 28 VOCs (Table 1) [11]. These compounds represented at least
5
five general classes of organic substance (acids, alcohols, esters, and lipids). All chemical
compounds, in this review, are described on the basis of their appropriate NIST data base
names since this base was used to provide preliminary identification information. Final
identification of the volatile compounds was done by GC/MS of authentic compounds
obtained from commercial sources or synthesized by us or others and compared directly
by GC/MS to the VOCs of the fungus (Table 1) [11]. With this chemical information in
hand, along with the DNA sequence data, we felt secure in proposing a binomial for this
fungus derived from the Latin –Muscodor (stinky) albus (white) [10].
Ultimately, artificial mixtures of the compounds were used in a biological assay system
to demonstrate the relative activity of individual compounds [11]. Although over 80% of
the volatiles could be identified, this seemed to be adequate to achieve an excellent
reproduction of the lethal- antibiotic effects of the VOCs that were being produced by the
fungus [11]. The bioassay test was used to examine the five general classes of VOCs.
Each class possessed some inhibitory activity with the esters being the most active (Table
2) [11]. Of these, the most active individual compound was 1-butanol, 3 methyl-, acetate.
However, it is to be strongly stressed that no individual compound or class of compounds
was lethal to any of the test microbes which consisted of representative plant pathogenic
fungi, Gram positive and Gram negative bacteria, and others [11]. Obviously, the
antibiotic effect of the VOCs of M. albus is strictly related to the synergistic activity of
the compounds in the gas phase (Table 2).
The mechanism of action of the VOCs of M. albus. on target fungi and bacteria is
unknown. However, a microarray study to analyze the transcriptional response of
6
Bacillus subtilis cells exposed to M. albus VOCs showed that genes involved in DNA
repair and replication were increased in expression (RA Britton, Michigan State
University, unpublished observations). These preliminary results would indicate that M.
albus VOCs are inducing some type of DNA damage in cells, possibly through the
effects of one of its naphthalene derivatives, since such related compounds had
previously shown to cause chromosome decondensation in bacteria [12]. Likewise,
similar microarray studies on other microorganisms susceptible to M. albus gases would
be helpful in elucidating the mechanism of antimicrobial action.
Using the bioassay test system it was possible to calculate the 50% inhibitory
concentration (IC50) for each test microbe and compare the killing ability of the artificial
mixture with that of the M. albus VOCs [11]. One of the most sensitive fungi was
Rhizoctonia solani and one of the least sensitive was Fusarium solani. In the later case,
F. solani is only inhibited by both the artificial VOC mixture as well as the VOCs of the
fungus. Finally, the viability of all test organisms was also determined after exposure to
either the fungus or to its artificial mixture of VOCs (Tables 1& 2) [11]. The artificial
mixture generally mimicked the effects of the fungus itself (Table 2) [11]. A minimum of
three VOCs (naphthalene, propanoic acid and butanol, 3-methyl) can quite effectively
mimic the killing activity of the fungal VOCs (13). Not all compounds in the fungal
VOCs are necessary for biological activity, but there may still be others that have gone
undetected that are critical to bioactivity [11].
7
Other isolates and species of Muscodor
Recently, seven new M. albus isolates were retrieved from the Northern Territory of
Australia [13]. These organisms were obtained by using M. albus (isolate 620) as a
selection tool in Petri plates containing agar in the presence of the plant tissues containing
endophytic fungi [13]. The host plants of these isolates were Terminalia prostrata
(Combretaceae), Kennedia nigriscans (Leguminosae) and Grevillea pterifolia
(Proteaceae). Each isolate was biologically active, produced some but not all of the
VOCs made by M. albus 620, and had between 96 % and 99% ITS-5.8S rDNA sequence
similarity to M. albus 620 [13].
Another isolate of M. albus has been recovered as an endophyte from nutmeg (Myristica
nutans- family Myristaceae) in Thailand [14]. This isolate has 98% sequence similarity to
the ITS-5.8 rDNA of M. albus and produces many of the same volatiles as isolate 620
and it also possesses antibiotic properties. Furthermore, again using the selection
technique, M. albus isolate 1-41-3s was obtained from an unidentified vine in the
Sumatran jungle of Tesso Nilo in Indonesia, and it possessed 98% ITS-5.8SrDNA
sequence similarity to M. albus 620 [15]. This isolate possesses unusual hyphae, a slime
layer, and some VOCs not observed before in other M. albus isolates including
aciphyllene, 2-butanone, 2-methyl furan, tetrahydrofuran and large amounts of an
unusual azulene derivative [15]. Discovery of these new isolates of M. albus confirms
that this organism may be a bona fide novel endophytic fungal genus rather than the
8
original 620 isolate occurring as a localized phenomenon in nature. The discovery also
shows that several plant families other than Lauraceae (Cinnamomum sp.) can serve as a
host for M. albus.
Two other species of Muscodor have been reported including M. roseus (endophytic on
Grevillea pteridifolia, Australia) and M. vitigenus (endophytic on Paullinia paullinoides,
Peru) [16,17]. These isolates are also closely genetically related (ITS-5.8S rDNA
sequences) to the Australian isolates of M. albus at the 96%-99% level [13]. The later
organism makes only naphthalene as a VOC and its repellency towards a plant associated
insect has been demonstrated [18].
Another isolate of M. albus (E-6) with unusual biochemical and biological properties, has
been obtained from the branches of a mature Guazuma ulmifolia (Sterculiaceae) tree
growing in a dry tropical forest in SW Ecuador [19]. This unique organism produces
many VOC’s not previously observed in other M. albus isolates including: butanoic acid,
2-methyl-; butanoic acid, 3-methyl-;2-butenal, 2-methyl-; butanoic acid, 3-methylbutyl
ester; 3-buten-1-ol, 3-methyl; guaiol; 1-octene, 3-ethyl-, formamide,N-(1-methylpropyl),
and along with certain azulene and naphthalene derivatives. On the other hand, some
compounds usually seen in M. albus isolates also appeared in the VOCs of isolate E-6
including: caryophyllene; phenylethyl alcohol; acetic acid, 2-phenylethyl ester;
bulnesene; and various propanoic acid, 2-methyl- derivatives. Scanning electron
micrographs of the mycelium showed substantial intertwining of the hyphal strands.
These strands seemed to be held together by an extracellular matrix accounting for the
strong mat-like nature of the mycelium which easily lifts off the agar surface upon
9
transfer unlike any other isolate of this fungus. The ITS -5.8S rDNA partial sequence
data showed 99% similarity to the original M. albus strain- 620. Now, for the first time, a
successful establishment of M. albus into its natural host, followed by recovery of the
fungus, was accomplished in seedlings of G. ulmifolia. The biological activity of the
VOCs of E-6 appears different from the original isolate of this fungus- 620 since a Gram
positive bacterium was killed and Sclerotinia sclerotiorum along with Rhizoctonia solani
were not.
Physiological aspects of VOC production
The composition of the medium greatly influences the quality and effectiveness of the
VOCs emitted by M. albus-620 [20]. For instance, a sucrose enriched medium primarily
yields methyl isobutylketone and acetic acid, butyl ester as the primary volatiles, yet
neither of these compounds appeared in any other medium. Furthermore, the VOCs under
these conditions, were limited in their bioactivity. More enriched media were more
effective in inhibiting a suite of plant pathogens used as test microbes [20].
Although only qualitative methodology was initially used to gather information on the
fungal VOCs there was a need to obtain quantitative data on VOC production. A
relatively new technology called proton transfer reaction-mass spectrometry (PTR-MS)
was used to monitor the concentration of VOCs emitted by M. albus [21]. PTR-MS is a
particularly useful technique because it can be run on line while continuously yielding
data on the concentrations of specific ions of interest. Data gathered from a long term M.
albus culture in a carboy by PTR-MS indicated that the production of VOCs is
10
temperature-dependent with diurnal fluctuations in gas production occurring as the
temperature varied [21]. Furthermore, continuous monitoring after 3 weeks revealed a
slow, but steady decline in VOC production which is probably a reflection of the
depletion of the carbohydrate sources in the potato dextrose agar. This is consistent with
the observations showing VOC production is related to the presence of a carbohydrate
source [20].
The PTR-MS technique was also applied to soils containing M. albus along with the plant
pathogen Pythium ultimum and it was possible to show the production of VOCs from M.
albus in situ. An estimation of the range of concentrations of total VOCs being produced
by M. albus is in the order of 100-300 ppb based on the determination of the
concentration of propanoic acid, 2-methyl, methyl ester.
Interestingly, since M. albus has not been artificially established in any host or non- host
plant, no data are available on the in planta production of VOCs by endophytically
established M. albus. This is an important phenomenon awaiting observation and the
PTR-MS technique seems like the most useful method to make such observations.
Establishing a successful M. albus/ plant relationship is critical in doing these in planta
mass spectral studies.
“Mycofumigation” with Muscodor spp.
The VOCs of M. albus kill many of the pathogens that affect plants, people and even
buildings [11,14](Table 2). The term “mycofumigation” has been applied to the practical
11
aspects of this fungus [11]. The first practical demonstration of its effects against a
pathogen was the mycofumigation of covered smut infected barley seeds for a few days.
The seeds were eventually planted and the resulting plants, in contrast to the untreated
control group, produced no infected heads [11]. Mycofumigation is also important for the
treatment of fruits in storage and transit [22]. Soil treatments have also been effectively
used in both field and greenhouse situations [23-25]. In these cases, soils are pretreated
with a M. albus formulation in order to preclude the development of infected seedlings.
M. albus is now being produced, by solid state fermentation, by the ton in order for its
use in many practical applications.
AgraQuest, an agricultural biotech company, of Davis, Calif., is developing M. albus for
numerous agricultural applications. The concept of mycofumigation, for a multitude of
uses, has the potential to replace hazardous substances that are currently applied to
humans, food, soil and buildings. The most notable of which is methyl bromide for soil
sterilization which use will soon cease because of its toxicity and negative influences on
the world’s ozone layer. On the other hand, it turns out that the VOCs of M. albus appear
safe, effective and environmentally friendly and may serve as a replacement soil
treatment [25].
Other VOC producing fungi
Using M. albus as a selection tool, Gliocladium sp. was isolated from Eucryphia
cordifolia in Patagonia and this organism is phylogenetically related to Trichoderma
spp.(Fig.2)[26]. It produces a series of VOC that have some bioactivity. Generally,
12
Trichoderma spp. although possessing VOCs with biological activity, are not sufficiently
active for practical applications to be made of them. However, in the case of
Gliocladium sp. at least two target organisms were killed by its VOCs including Pythium
ultimum and Verticillum dahliae [26].
Concluding remarks and future perspectives
Obviously, because of the impressive biological activity of M. albus and its introduction
into practical agriculture/industry, it seems that the fungus should be more fully studied
relative to its location and role in nature. Overall, the most pressing question regarding
M. albus is the mode of action of a multitude of volatile compounds and how they act
synergistically to cause the death of microbes.
Certainly, knowledge of its hosts and those factors controlling host preference may
eventually allow for the use and development of this organism for hosts that it does not
naturally frequent. Such information may result in still more applications and direct uses
of M. albus. Further, we need to learn if Muscodor isolates can be directly inoculated into
agricultural and forest species in order to provide protection against invading pests and
pathogens. In this regard, it has been recently learned that M. albus as well as a mixture
of its VOCs has activity against certain plant pathogenic nematodes [27]. Other studies
are in progress to test its biological activity against plant insect pests.
It seems that there may be some factors that may limit the use of M. albus for biological
control. One of the most important of which is the inability of M. albus to kill, and only
13
inhibit isolates of Fusarium spp. This seems to be a universal observation with all
isolates of Muscodor sp. [11,13,15]. The fusarial species are some of the most important
plant pathogens occurring on a wide range of agricultural and forest species. Perhaps,
other Muscodor spp. will be found in places that have not yet been sampled including
such locations as Australia, Brazil, Malaysia, USA, Venezuela, and tropical Africa that
will be effective against Fusarium spp. Furthermore, unless adequate sources of
carbohydrates are available, M. albus ceases producing its VOCs [20]. This may be
present a problem in situations in which VOC production stops even though unwanted
organisms persist.
In addition, it is extremely important to possess information on the distribution, life cycle
and other aspects of the chemistry of this organism. This will aid in expanding its utility
as a biological control agent. Hopefully, the development of information on M. albus
will have broad implications for the discovery and development of other rainforest
microbes. Work on this important fungal genus has just begun.
Acknowledgements
The author acknowledges the financial assistance of the NSF, the Montana Board of
Research and Commercialization Technology, the USDA, and the Montana Agricultural
Experiment Station in making this work possible.
14
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17
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18
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19
Table 1. GC/MS analysis of the volatile compounds produced by M. albus.
Several minor peaks and the breakthrough peak were omitted from the total analysis since
they represent only 1% of the total area. Compounds found in the control PDA plate
are not included in this table [11].
RT
Total
m/z
Possible compound
MW
Area
(%)
3:45
0.33
114
Octane
114
4:19
0.93
58
Acetone
58
74
4:37
0.68
74
Methyl acetate
5:56
7.63
88
Ethyl acetate
88
0.31
102
Propanoic acid, 2-methyl, methyl ester
102
6:51
7:16
6.24
*
Ethanol
46
8:03
2.07
116
Propanoic acid, 2-methyl-ethyl ester
116
144
11:45
0.58
*
Propanoic acid, 2-methyl 2methylpropyl ester
12:05
2.06
74
1-Propanol, 2-methyl74
12:50
22.24
*
1-Butanol, 3-methyl, acetate
130
158
14:57
1.53
*
Propanoic acid, 2-methyl, 3methylbutyl ester
15:28
22.99
*
1-Butanol, 3-methyl88
16:08
0.29
138
#Furan, 2-pentyl138
18:53
0.29
142
#4-Nonanone
142
20:38
0.41
142
2-Nonanone
142
204
21:07
0.30
204
# Naphthalene, decahydro-4a-methyl-1methylene-7-(1-methylethylidene)-,
(4aR-trans)22:54
1.51
204
# Azulene, 1,2,3,4,5,6,7,8-octahydro204
1,4-dimethyl-7-(1-methylethenyl)-,[1S(1.alpha.,4.alpha.,7.alpha.)]
23:16
0.94
204
# Cyclohexene, 4-(1,5-dimethyl-1,4204
hexadienyl)-1-methyl25:20
3.63
204
# 1H-3a,7-methanoazulene,
204
2,3,4,7,8,8a-hexahydro-3,6,8,8
tetramethyl-, [3R-(3.alpha.,
3a.beta.,7.beta.,8a.alpha.)]
25:30
6.08
88
Propanoic acid, 2-methyl
88
26:04
0.48
204
Caryophyllene
204
204
27:55
0.34
204
# Naphthalene,1,2,4a,5,6,8a-hexahydro4,7-dimethyl-1-(1-methylethyl)-, [1R(1.alpha., 4a.alpha.,8a.alpha.)]
28:34
0.36
204
# Spiro[5.5]undec-2-ene,3,7,7204
trimethyl-11-methylene28:50
1.07
204
Azulene, 1,2,3,5,6,7,8, 8a-octahydro-1,
204
4-dimethyl-7- (1-methylethyenyl)-, [1S(1.alpha.,7.alpha.,8a.beta.)]
Common Name: Bulnesene
204
28:57
3.24
204
Naphthalene, 1,2,3,5,6,7,8,8aoctahydro-1,8a-dimethyl-7-(1-
20
31:12
33:17
39:00
1.74
1.06
9.76
*
122
204
methylethenyl)-,[1R(1.alpha.,7.beta.,8a.alpha.)]
Common Name: Valencene
Acetic acid,2-phenylethyl ester
Phenylethyl alcohol
# Unknown
164
122
204
* No molecular-ion peak was observed in the spectrum of either the standard compound or the compound
undergoing the analysis.
# Denotes that a spectrum and retention time of this component was observed and the substance matched to
the most likely compound in the NIST data base, but the data have not been confirmed by use of an
appropriate identical standard compound by either retention time or MS. These compounds were not placed
in the artificial mixture in the bioassay test.
21
Table 2. The effects of the volatile compounds of M. albus and an artificial mixture of M. albus
compounds on a group of test fungi and bacteria. After exposure to M. albus gasses, the test organism was
evaluated for its growth and viability after removal from the gases. The artificial atmosphere consisted of
the compounds identified after analysis of the M. albus gases (Table 2)*. The growth of the test organisms
in the artificial atmosphere was measured after exposure to the artificial mixture of compounds at 3.290μl/50CC in order to obtain IC50’s. The % growth over the control and viability were measured after
exposure to 60μl/50CC. Viability was determined after the removal of the compounds at 3 days [11].
Test Microbe
Pythium ultimum
Phytophthora cinnamoni
Rhizoctonia solani
Ustilago hordei
Stagnospora nodorum
Sclerotinia sclerotiorum
Aspergillus fumigatus
Fusarium solani
Verticillum dahliae
Cercospora beticola
Tapesia yallundae
Xylaria sp.
Muscodor albus
Escherichia coli
Staphlococcus aureus
Micrococcus luteus
Candida albicans
Bacillus subtilus
% Growth
over control
after a 2 day
exposure to
M.albus
0
0
0
0
0
Viability
after 3 days
exposure to
M. albus
culture
Dead
Dead
Dead
Dead
Dead
IC50 in
artificial
atmosphere
for 2days
(μl/CC)
0.48±0.01
0.29±0.06
0.08±0.02
0.31±0.09
0.15±0
% Growth
(mm) over
control in
artifical
atmosphere
0
0
0
0
0
Viability
after 3 days
exposure
artificial
atmosphere
Dead
Dead
Dead
Dead
Dead
0
0
19.4±0.28
0
17.5± 3.5
Dead
Dead
Alive
Dead
Alive
0.17±0.05
0.41±0.05
1.13±0.07
0.3±0
0.12±0.15
0
0
42.0 ±2
0
8±2
Alive
Alive
Alive
Dead
Alive
0
25±0
100±0
0
0
0
0
0
Dead
Alive
Alive
Dead
Dead
Dead
Dead
Alive
0.64±0
0.41±0.03
0.6±0
#
#
#
#
#
0
0
17.5±7.5
0
0
0
trace
0
Dead
Alive
Alive
Dead
Dead
Dead
Alive
Alive
* The amount of each positively identified compound used in the artificial mixture was obtained by
applying the electron ionization cross section (% of the total area) of the compound obtained in the GC/MS
analysis (Table 1). The artifical mixtures were subsequently tested by placing them in a presterilized
microcup (4x6 mm) located in the center of a test Petri plate containing PDA. Agar plugs containing
freshly growing test microbes (or streaked microbes) were positioned about 2-3 cm from the center
microcup. Then the plate was wrapped with 2 layers of parafilm and incubated for two or more days at
23°C. Measurements of linear mycelial growth were made from the edge of the inoculum agar plug to the
edge of the mycelial colony.
#Not measured in this experimental design.
22
23